Video 1 - 2D gel electrophoresis

[What is the purpose of this video?]

[The purpose of this video is to demonstrate how to run good, reproducible 2D gels, providing tips, techniques, and tricks to achieve high-quality results.]

[What are the three segments of the video?]

[The video is divided into three segments: 1) How to apply sample and rehydrate the IPG strip, 2) How to equilibrate the IPG strip in preparation for the second dimension gel, and 3) Staining of the second dimension gel.]

[What is the first step in running a 2D gel?]

[The first step is to rehydrate the IPG strip using a rehydration buffer and sample preparation.]

[What is the Bio-Rad 2D Starter Kit used for?]

[The Bio-Rad 2D Starter Kit is a tool for learning 2D gel electrophoresis, containing all the reagents and an E. coli sample needed to run a 2D gel.]

[What are the components of the rehydration buffer?]

[The rehydration buffer contains Urea, CHAPS, DTT, Ampholytes, and Bromophenol Blue.]

[How much E. coli sample is loaded onto the IPG strip?]

[200 micrograms of E. coli sample, reconstituted in rehydration buffer, is loaded onto an 11 cm pH 4-7 IPG strip.]

[How is the sample loaded into the focusing tray?]

[The sample is dispensed in a nice, even bead along one edge of the lane in the focusing tray, ensuring even distribution.]

[How is the IPG strip placed into the focusing tray?]

[The plastic protective backing is removed, and the gel side is placed face down onto the sample bead in the focusing tray, ensuring no air bubbles are trapped.]

[What is the purpose of mineral oil in the process?]

[Mineral oil is used to cover the IPG strips to prevent them from drying out during rehydration and the run.]

[What are the typical rehydration and focusing times?]

[Typical rehydration time is 12 hours, and focusing time ranges from 6 to 8 hours.]

[What is the equilibration process for the IPG strip?]

[The equilibration process is a two-step procedure using equilibration buffers: Buffer 1 contains DTT for protein reduction, and Buffer 2 contains iodoacetamide for protein alkylation.]

[How is the IPG strip prepared for the second dimension gel?]

[After equilibration, the IPG strip is rinsed in running buffer, placed gel side up on the gel cassette, and sealed with agarose overlay to prepare for the second dimension gel.]

[What is the purpose of the Precision Plus Standard Plug?]

[The Precision Plus Standard Plug is used as a broad-range standard for reference during the second dimension gel run.]

[How is the second dimension gel run?]

[The gel is run in a Criterion gel box at 200 volts for 1 hour, or until the dye front reaches the bottom of the gel.]

[What are the steps for staining the second dimension gel?]

[After running, the gel is rinsed three times in water to remove SDS, then stained with Bio-Safe Coomassie stain for 45 minutes to 1 hour, followed by destaining in water until the desired background is achieved.]

[What staining options does Bio-Rad offer for 2D gels?]

[Bio-Rad offers visual stains like Coomassie and silver stains, as well as fluorescent stains like Flamingo and SYPRO Ruby.]

[What is the final step after staining the gel?]

[The final step is imaging the gel, which should show a clear background with well-resolved protein spot